Substrate binding mutants of the higher plant ADP-glucose pyrophosphorylase.
To explore the structure-function relationships of the heterotetrameric higher plant ADP-glucose pyrophosphorylase, composed of a pair of large and small subunits, the small subunit cDNA was subjected to chemical mutagenesis and then co-expressed with the wild-type large subunit cDNA. Mutants were selected for their inability to complement a defective bacterial ADP-glucose pyrophosphorylase gene and, in turn, to accumulate glycogen as viewed by iodine staining of the cells. Based on these initial analyses, we subsequently identified four distinct classes of mutations which were glycogen-deficient but exhibited enzyme activity levels comparable to the normal recombinant enzyme under saturating reaction conditions. Three classes, each a product of single amino acid substitution, showed altered kinetic constants for substrates. Substitution of Asp252 to Asn conferred the enzyme lower affinity for glucose-1-phosphate, replacement of Asp121 to Asn resulted in an enzyme less responsive to both glucose-1-phosphate and ATP, while the Ala106 to Thr substituted enzyme contains altered sensitivity primarily to ATP. The fourth class, a Pro43 to Ser substitution, resulted in an enzyme with decreased sensitivity (8-fold) to the activator 3-PGA. Overall, the results of this study suggests that the two subunit types do not have identical roles in enzyme function and that the small subunit plays a more dominant role in catalysis than the large subunit.
Laughlin
M J
MJ
Genetics and Cell Biology, Washington State University, Pullman 99164, USA.
Payne
J W
JW
Okita
T W
TW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase